Ab6326

Bromodeoxyuridine (BrdU) was purchased from Sigma-Aldrich (St. Louis, MO, USA). AG490 was purchased from Calbiochem (San Diego, CA, USA). The following primary antibodies were used in these experiments: rat monoclonal anti-BrdU (Abcam, ab6326, 1:500), mouse anti-Sox2 (Santa Cruz, sc365823, 1:200), rabbit anti-Ki67 (Millipore, AB9260, 1:200), mouse anti-nestin (Abcam, ab6142, 1:200), rabbit anti-doublecortin (DCX) (Abcam, ab18723, 1:400), rabbit anti-glial fibrillary acidic protein (GFAP) (DAKO, 1:1500), rabbit anti-NeuN (Abcam, ab177487, 1:500), rabbit anti-Phospho-Stat3 (Tyr705) (Cell Signaling Technology #9145, 1:2000), and mouse anti-GAPDH (Abcam, ab8245, 1:10000). All secondary antibodies for immunofluorescent study were Alexa Fluor antibodies from Thermo Fisher Scientific (Waltham, MA, USA), namely goat anti-rat 488, goat anti-mouse 568, and goat anti-rabbit TRITC (T2769). All secondary antibodies for Western-blot were ECL™ anti-rabbit and anti-mouse IgG from GE Healthcare (UK) Company.

Mice were sacrificed, and tissues obtained were fixed in 4% paraformaldehyde overnight at 4°C. Dehydration of the samples was performed in a graded series of ethanol concentrations and xylene before they were embedded in paraffin wax. Sections of 10 μm thickness were obtained using a microtome and left overnight to dry at room temperature. Samples were deparaffinised in xylene and rehydrated in a graded series of ethanol solutions. Antigen retrieval was done in a microwave using 10 mM citrate buffer and for BrdU immunohistochemistry, samples were also incubated for 20 min in 2 M HCl. Blocking and antibody incubations were variable and optimal for each different antibody used. Primary antibodies used include: 1:100 BrdU (Abcam, ab6326, rat monoclonal), 1:500 K15 (SantaCruz, sc-47697, mouse monoclonal), 1:100 PCNA (SantaCruz, sc-25280, mouse monoclonal). Following primary antibody incubation samples were washed in PBS. The following secondary antibodies were used: Cy3-streptavidin, biotin-rat and biotin -mouse all from Jackson ImmunoResearch and also Vectastain universal secondary (Vector laboratories, #016-160-084). All images were acquired using a Zeiss Axio Observer.A1 microscope. Quantification was performed in a blinded fashion.

For immunofluorescence analysis, the following primary antibodies were used: mouse anti-ARL13B (1:1,000 dilution, Abcam, #Ab136648), Goat anti-IBA1 (1:500, Abcam, #Ab5076), mouse anti-IB4 (1:400, Vector Laboratories, #B-1205), rabbit anti-GFAP (1:3,000, Dako, #Z0334), rabbit anti-S100β (1:1,000, Proteintech, #15146-1-AP), rat anti-BrdU (1:1,000, Abcam, #ab6326), rabbit anti-CUX1 (1:500, Santa Cruz Biotechnology, #sc-13024), rat anti-CTIP2 (1:500, Abcam, #ab18465), rabbit anti-RSPH9 (kindly gifted from Zhu Xueliang, Shanghai Institute of Biochemistry and Cell Biology, CAS), rabbit anti-RSPH3 (1:1,000, Proteintech, #17603-1-AP).